凝血酶体外诱导模拟脑室出血后大鼠蛛网膜细胞纤维化的实验研究

目的 观察凝血酶（thrombin）刺激体外培养大鼠蛛网膜细胞后纤维化程度的变化，探讨其与脑室出血（intraventricular hemorrhage，IVH）后大鼠慢性脑积水形成的关系。方法 采用SD大鼠蛛网膜细胞，经不同浓度凝血酶刺激，在体外模拟脑室出血（IVH）后慢性脑积水大鼠的蛛网膜细胞纤维化的病理生理过程。细胞免疫化学法（immunocytochemistry ，ICC）鉴定传代培养的SD大鼠蛛网膜细胞标志物细胞角蛋白（CK 8/18）和桥粒蛋白（Desmoplakin）的表达。qRT-PCR与Western Blot检测细胞纤维化因子（Col-I、Col-III、TGF-β1、α-SMA）的表达，观察其纤维化程度，同时检测比较各组细胞的表达差异。结果 （1）培养的蛛网膜细胞标志物Cytokeratin和Desmoplakin表达阳性。（2）凝血酶可诱导蛛网膜细胞纤维化，在50u/ml浓度下，蛛网膜细胞纤维化程度最明显。（3）实验组较对照组细胞纤维化程度明显。结论 体外可以稳定培养大鼠蛛网膜细胞。凝血酶诱导的蛛网膜细胞各种纤维化指标明显高于正常组，提示纤维化模型构建成功。构建成功的纤维化蛛网膜细胞有利于下一步深入研究脑室出血后慢性脑积水的机制。     

动脉瘤性蛛网膜下腔出血后慢性脑积水的研究进展

动脉瘤性蛛网膜下腔出血后慢性脑积水是动脉瘤破裂后蛛网膜下腔出血的晚期并发症之一,可加重病情,延长病程,造成神经功能障碍。其发生机制可能与蛛网膜下腔出血后蛛网膜下纤维化有关。影响该病发生的临床因素复杂,目前还没有满意的防治措施。进一步明确其发病机制和临床危险因素,进而能够有效地预防本病的发生对改善动脉瘤性蛛网膜下腔出血的预后有重要意义。     

大鼠实验性脑室出血后LN及LNR-1在脑内的表达及意义

目的观察大鼠脑室出血后层粘蛋白（LN）及其受体LNR-1在大鼠脑内的表达变化，探讨其与出血后慢性脑积水之间的关系。方法将大鼠随机分为3组：正常对照组、假手术对照组、实验组，将枸橼酸化的0．1ml自体静脉血注入实验组大鼠侧脑室内建立出血后慢性脑积水模型（假手术组注入0．1ml生理盐水），采用免疫组化方法观察出血后不同时间LN及LNR-1在脑内的表达变化。结果实验组30d出现脑积水为80％（4／5）、其余组别未出现慢性脑积水；实验组出血后LN在纵裂池、软脑膜、蛛网膜、硬膜、脑内毛细血管壁、蛛网膜下腔血管壁的表达在3d时增加，7d表达继续增加，14d表达最强，较对照组、假手术组显著增强（P〈0．01），30d仍强烈表达；实验组出血后LNR-1在脉络丛的立方上皮细胞胞浆及顶质膜、室管膜细胞、软脑膜细胞、蛛网膜细胞、硬膜、纵裂池、胶质细胞的表达增加。LNR-1在蛛网膜下腔的表达变化与LN基本一致。结论大鼠脑室出血后LN及LNR-1的表达上调可能参与蛛网膜纤维化的过程，从而在出血后慢性脑积水的形成中发挥重要作用。     

大鼠蛛网膜下腔出血后脑血管储备的实验研究

目的探讨大鼠蛛网膜下腔出血(SAH)模型不同时期脑血管储备功能的变化。方法将120只SD大鼠分为空白对照组、盐水对照组、盐水+乙酰唑胺(ACZ)组、SAH模型组和SAH+ACZ组,其中盐水对照组、盐水+ACZ组、SAH模型组和SAH+ACZ组又分为1 d、3 d、5 d、7 d 4个亚组,对各亚组大鼠进行神经行为学评分,测定不同时间大鼠皮层脑血流。结果 SAH模型组脑血流较空白对照组及盐水对照组有不同程度的下降,差异具有统计学意义(P0.05);盐水对照组与盐水+ACZ组之间的脑血流变化及神经行为学评分比较无统计学差异(P0.05);SAH+ACZ组与SAH模型组比较脑血流及神经行为学评分均下降,以5 d组为著。结论 SAH后不同时期脑血管储备功能及神经行为学评分均降低,且二者具有相关性。     

动脉瘤性蛛网膜下腔出血后的脑积水

动脉瘤性蛛网膜下腔出血后的脑积水杨俊，苏亦兵，赵继宗我院从１９８２年至１９９２年，收治了因颅内动脉瘤破裂出血所致急性脑室扩张、脑积水病人２３例，约占同期颅内动脉瘤的０．４％，现报告如下。临床资料１．一般资料：男１２例，女１１例，年龄２４～６７岁，平均...     

动脉瘤性蛛网膜下腔出血后慢性脑积水

目的探讨动脉瘤性蛛网膜下腔出血后(aSAH)慢性脑积水的发生率及其易患因素。方法回顾性研究2003年1月至2005年9月我科aSAH病例221例,采用单因素及多因素统计方法分析与慢性脑积水发生的相关影响因素。结果慢性脑积水的发生率为12.7%(28/221)。经单因素分析显示,患者年龄、Hunt-Hess级别、Fisher级别、前交通动脉瘤、aSAH次数以及脑室内出血具有统计学意义;多因素logistic回归分析显示,Fisher级别、脑室内出血、aSAH次数、前交通动脉瘤具有统计学意义。结论慢性脑积水为aSAH后一较常见并发症,影响其发生的高危因素包括Fisher级别、脑室内出血、aSAH次数、前交通动脉瘤。对具上述危险因素的aSAH患者应注意跟踪随访,及时诊治。     

猪实验性脑室出血后慢性脑积水模型的建立及蛛网膜下腔脑脊液循环的改变

目的建立猪实验性脑室出血后慢性脑积水动物模型,对脑出血后慢性脑积水的发生机制进行初步研究。方法雌性贵州小型猪20只,健康对照组5只、实验组15只。实验组分别将15ml(1只)、12ml(1只)、8ml(13只)枸橼酸化的自体静脉血注入侧脑室内建立猪实验性脑室出血后慢性脑积水动物模型;健康对照组不作处理。采用MRI动态观察脑室变化,SPECT观察蛛网膜下腔CSF循环的改变,观察病理及超微结构改变。结果健康对照组死亡1只(1/5)、实验组死亡4只(4/15)。实验组脑室出血后30d有7只(7/11)形成慢性脑积水;健康对照组未出现脑积水。脑室出血后各时相点蛛网膜下腔核素显影均局限在椎管穿刺点附近,显示CSF循环障碍持久存在;脑室出血后蛛网膜下腔轻度增宽、蛛网膜细胞间隙纤维样结构增多,未形成脑积水动物的病理改变较形成脑积水者轻。结论猪脑室出血后蛛网膜下腔的CSF循环吸收障碍可能与电镜所发现的蛛网膜纤维化有关;蛛网膜纤维化可能是出血后慢性脑积水的主要形成原因之一。     

血管平滑肌细胞凋亡在大鼠蛛网膜下腔出血后血管痉挛中的表达

目的：研究血管平滑肌细胞凋亡在大鼠蛛网膜下腔出血（ SAH ）后脑血管痉挛中的表达。方法采用枕大池二次注血法建模,42只SD大鼠随机分成正常对照组、假手术组、实验组。根据取材时间不同,实验组分为SAH后1 d、3 d、5 d、7 d及14 d 5个组。观察基底动脉病理改变,TUNEL法检测平滑肌细胞凋亡变化。结果实验组细胞凋亡阳性细胞数以第1d表达增高,第3d、5d仍持续高位表达,第7d后降低,第14 d时明显下降,实验组平滑肌细胞凋亡与正常对照组比较有显著差异（14 d组除外）（均P﹤0．01）。结论血管平滑肌细胞凋亡在大鼠蛛网膜下腔出血后血管表达明显增高,血管平滑肌细胞凋亡在脑血管痉挛发病机制中可能存在一定作用。     

实验性蛛网膜下腔出血后柔脑膜纤维化的研究

目的 研究实验性蛛网膜下腔出血后柔脑膜的纤维化及其观测指标.方法采用SD大鼠枕大池两次注血法建立模型,分别在3d、6d、10d、14d、21d取脑脊液检测转化生长因子-β1(TGF-β1)和Ⅰ型前胶原羧基端前肽(PICP);21d的脑组织予以冰冻切片后,行MASSON染色观察柔脑膜的胶原纤维.结果 (1)实验组脑脊液中的TGF-β1较对照组明显升高并呈双时相(P<0.01).21d柔脑膜胶原纤维较对照组增多(P<0.01);(2)实验组脑脊液中的PICP较对照组明显升高(P<0.01);(3)21d PICP与柔脑膜的胶原纤维衡量指标显著正相关(r=0.605,P<0.05).结论 SAH发生后,可引起TGF-β1的升高,柔脑膜纤维化;脑脊液中PICP可作为检测柔脑膜的纤维化程度的指标.     

蛛网膜下腔出血继发早期脑室扩张积水：附69例报告

本文观察了98例 SAH 病人,发现69例有早期脑室扩张积水,并对其中34例进行了 CT 复查。结果:SAH 后继发早期脑室扩张积水是较为常见的现象,其发生率在70%左右。脑室扩张积水与颅内积血、病情程度及死亡率有关。颅内积血的吸收速度及 SAH 再发生和脑室扩张的转归有关。脑室扩张积水的程度变化是预测病人生存率的敏感指标之一。     

Posttraumatic intraventricular arachnoid cyst accompanied by pseudomeningoencephalocele in a child

Growing skull fracture (GSF) is a rare complication of head trauma. A posttraumatic intraventricular arachnoid cyst (AC), neither isolated nor accompanied by a GSF has not been reported previously. A seven-year-old girl was admitted after a severe head injury with a separated right parieto-occipital fracture and contusion. She responded well to conservative therapy. Seven weeks after discharge, she was re-admitted with a large parieto-occipital pseudomeningoencephalocele due to herniation of cerebrospinal fluid and neural tissue to the subgaleal space through the widened fracture defect, an extra-axial cyst at the posterior interhemispheric space and an intraventricular cystic mass. She underwent open surgery, and the intraventricular cystic mass was totally removed. The histological findings were consistent with an AC. One week after dural repair, hydrocephalus developed, and a ventriculo-peritoneal shunt was inserted. She did well during two-year follow-up. The present case is unique as an intraventricular AC following head trauma. When an intraventricular cystic lesion is encountered after severe head trauma, the possibility of an AC should be considered; especially with neighboring contused neural tissue and leptomeningeal cyst formation.     

蛛网膜细胞与血性CSF共同培养后HLA-DR的表达变化

目的研究蛛网膜细胞(Ar细胞)与血性脑脊液(CSF)共同培养后人类白细胞抗原- DR(HLA-DR)的表达变化。方法(1)Ar细胞的培养、鉴定和HLA-DR基础表达的观察。(2)采用Ar细胞和血性CSF共同培养的方法,用流式细胞仪(FCM)和免疫荧光染色观察Ar细胞HLA-DR的表达改变。结果95%以上培养的Ar细胞均有HLA-DR的基础表达。在血性CSF刺激下,Ar细胞HLA-DR的表达明显增强,免疫荧光法和FCM检测出了一致的时间表达变化模式,在1-5d呈基础表达状态;6d左右表达开始升高;7d、8d表达显著增强,此时荧光颗粒数量和强度最高,阳性细胞数可达61.2%;第9天后表达逐渐降低,阳性细胞数减少,第10天时减低至12.5%,荧光数量和强度亦明显降低。结论在血性CSF刺激下,Ar细胞通过HLA-DR表达增高,可以发挥抗体提呈细胞(APC)的功能,提示其可能与SAH并发症尤其是迟发性脑缺血(DCI)的发生机制相关。     

Cell growth of immortalized arachnoid cells in the presence of fibroblasts and blood products

ObjectThe pathophysiology of non-obstructive hydrocephalus involves alteration in cerebrospinal fluid (CSF) pathways. The exact mechanism is unknown, but as arachnoid CSF egress is a major route of CSF removal, damage or alteration to the growth of arachnoid cells may influence the rate of CSF absorption. We investigated the effect of soluble factors secreted by fibroblasts and the presence of blood products on arachnoid cell growth.MethodsAn immortalized arachnoid cell line was developed and cells were grown on semipermeable membranes in a culture chamber. Arachnoid cells were plated in Transwells®, with fibroblasts separated from the arachnoid cells. Cell phenotype was analyzed and cell growth rates were determined by manual counts. Similar experiments were conducted with biliverdin, bilirubin, as well as fibroblast challenge. DNA content in the cell cultures was then determined as corroborative data. Cell counts for the additional arachnoid cell lines were calculated at each day and represented the controls.ResultsCell counts increased with each time point. Arachnoid cells in the three experimental conditions showed a statistically significant decrease in cell counts for each day when compared to the control group. Post hoc analysis showed differences between the control and experimental conditions but no significant difference between groups. The DNA content for each experimental condition was reduced at all time points when compared to the control arachnoid cells, but only became statistically significant at day 7.ConclusionInflammation and hemorrhage are two common conditions associated with the development of hydrocephalus. The arachnoid membrane is exposed to fibroblasts and blood products (bilirubin, biliverdin) in these conditions, and their effect on arachnoid cell growth was studied. We have shown that arachnoid cell growth decreases in the presence of fibroblasts, bilirubin, and biliverdin. Given its intimate relationship with CSF, it is possible that the decreased growth of arachnoid cells may affect absorption and thus contribute to the development of hydrocephalus.     

Effect of thrombin preconditioning on migration of subventricular zone-derived cells after intracerebral hemorrhage in rats

Objective: To investigate the effect of thrombin preconditioning (TPC) on the intracerebral hemorrhage (ICH)-induced proliferation, migration, and function of subventriclular zone (SVZ) cells and to find new strategies that enhance endogenous neurogenesis after ICH.

Methods: Male Sprague-Dawley rats were randomly divided into 3 groups (ICH, TPC, and control group). Rats of each group were randomly divided into 5 subgroups (3-d, 7-d, 14-d, 21-d, and 28-d subgroup). ICH was caused by intrastrial stereotactic administration of collagenase type IV. Brdu was used to label newborn SVZ cells. Organotypic brain slices were cultured to dynamically observe the migration of SVZ cells at living brain tissue. Migration of Dil-labeled SVZ cells in living brain slices was traced by time-lapse microscopy. To assess whether SVZ cells migrating to injured striatum had the ability to form synapses with other cells, brain slices from each group were double immunolabeled with Brdu and synapsin I.

Results: The number of Brdu-positive cells markedly increased in the ipsilateral SVZ and striatum 3 days after TPC, peaked at 14 days (P < 0.01), continued to 21 days, and then gradually decreased at 28 days with significant difference compared to the ICH group at each time point (P < 0.01). Migration of Dil-labeled SVZ cells in brain slices in each group was observed and imaged during a 12-h period. Dil-labeled SVZ cells in the TPC group were observed to migrate laterally toward striatum with time with a faster velocity compared to the ICH group (P < 0.01). Our study also demonstrated that TPC induced strong colocalization of Brdu and synapsin I in the ipsilateral striatum between 3 and 28 days after injury.TPC made colocalization of Brdu and synapsin I appear earlier and continue for a longer time compared to the ICH group.

Conclusions: Our results demonstrated that TPC could promote proliferation, migration, and function of SVZ cells after ICH, which may provide a new idea for enhancing endogenous neurogenesis and developing new therapeutic strategies against ICH-induced brain injury.     

凝血酶预处理对大鼠脑出血后内源性神经再生的影响

目的 探讨凝血酶预处理(thrombin preconditioning,TPC)对大鼠脑出血后内源性的神经细胞再生的影响。方法 SD大鼠随机分为对照组、脑出血(Intracerebral hemorrhage,ICH)组和 TPC组,每组分为3、7、14、21、28 d亚组。应用胶原酶脑内立体定向注射制作脑出血模型。TPC组首先将1U凝血酶立体定向注入右侧纹状体,1 d后再制作脑出血模型。所有大鼠应用BrdU腹腔注射在体标记再生的脑室下带( subventricular zone, SVZ)细胞,应用免疫组织化学技术检测BrdU阳性细胞。结果 脑出血后3 d同侧SVZ 和基底节BrdU阳性细胞开始增加,但与对照组比较差异没有显著性(P>0.05),7 d时BrdU阳性细胞数增加明显,与对照组比较有明显差异(P<0.05),14 d达到高峰,然后逐渐下降,28 d时BrdU阳性细胞虽然仍有增加,但与对照组比较差异没有显著性(P>0.05)。TPC组3 d时BrdU阳性细胞数目开始增加,与脑出血组和对照组比较,差异均具有显著性(P<0.01),14 d达高峰,一直持续至21 d,28 d时BrdU阳性细胞虽然略有下降,但与对照组和脑出血组比较,仍有统计学差异(P<0.05)。结论 凝血酶预处理能够增强脑出血后内源性神经再生,可能为脑出血后内源性神经再生的研究提供新的思路。     

凝血酶预处理对大鼠脑出血后SVZ细胞迁移和分化的影响

目的 探讨凝血酶预处理(thrombin preconditioning,TPC)对大鼠脑出血(intracerebral hemorrhage,ICH)后侧脑室下带(subventricular zone,SVZ)细胞迁移和分化的影响。方法 将90只SD雄性大鼠随机分为3组:TPC组、ICH组、对照组。每组分为3、7、14、21、28 d亚组; 应用IV型胶原酶脑内立体定向注射制作脑出血模型,应用Brdu标记新生的SVZ细胞; 进行脑片培养,应用时间间隔显微系统动态观察Dil标记的SVZ细胞在活体脑片上的迁移; 为了评估迁移至损伤区的SVZ细胞是否具有与其他细胞形成突触的能力,进行Brdu和突触蛋白Ⅰ免疫组织化学双标染色。结果 在时间间隔显微系统下观察Dil标记的SVZ细胞向纹状体迁移,动态观察12 h,TPC组3 d SVZ细胞迁移的速度是(4.23±0.25)μm/h,7 d的速度是(6.53±0.37)μm/h,14 d的速度是(8.23±0.47)μm/h,21 d的速度是(7.05±0.31)μm/h,28 d的速度是(5.29±0.20)μm/h,在各个时间点TPC组的迁移速度明显快于脑出血组(P<0.01)。TPC组同侧纹状体Brdu和突触蛋白Ⅰ双标阳性细胞3 d明显增加,14 d达高峰,持续至21 d,然后逐渐减少。脑出血组3 d未见Brdu和突触蛋白Ⅰ双标阳性细胞,7 d可见少数阳性细胞,14 d达高峰,然后逐渐下降,在各个时间点双标阳性细胞与脑出血组比较均明显增加(P<0.01)。TPC使突触蛋白I的表达出现时间更早,持续时间更长。结论 TPC能够促进脑出血后SVZ细胞的迁移和分化。     

去铁敏对大鼠脑室出血后慢性脑积水与脑组织Wnt1、Wnt3a表达的影响

目的 观察去铁敏对大鼠脑室出血(IVH)后脑积水的干预效果及脑组织Wnt1、Wnt3a的表达影响.方法 雄性SD大鼠130只随机分为正常组、假性IVH组、IVH组及IVH加去铁敏组.采用脑室出血后慢性脑积水大鼠模型,于术后1 d、7 d及28 d处死大鼠,观察脑积水的发生率和脑组织中Wnt基因及蛋白表达情况.结果 正常组、假性IVH组未见脑积水发生,IVH组28 d脑积水发生率80%(12/15),显著高于IVH加去铁敏组28 d脑积水发生率20%(3/15).IVH后Wnt1、Wnt3amRNA及蛋白表达在7 d及28 d均显著高于假性IVH组,而去铁敏治疗组Wnt1、Wnt3a mRNA及蛋白表达在28 d较IVH组均显著降低.结论 去铁敏治疗可降低大鼠IVH后慢性脑积水发生率,Wnt信号通路参与了IVH后脑积水发生及去铁敏干预机制.

Abstract：

Objective To observe the effect of deferoxamine on chronic hydrocephalus after intraventricular hemorrhage (IVH) and the role of Wnt (Wnt1 and Wnt3a). Methods A total of 130Sprague Dawley male rats were randomly assigned into 4 groups: normal control group, sham - IVH group,IVH group and deferoxamine - treated group. The rats of subgroups except normal control group received an injection of autologous blood or saline into right lateral cerebral ventricles. Deferoxamine or vehicle was administered 3 hours after IVH and then every 12 hours up to 7 days in IVH group and deferoxaminetreated group. Rats were euthanized at 1, 7, 28 days for measurement, and the transverse diameter of the lateral ventricles were observed for evaluation of hydrocephalus. The expression of Wnt1 and Wnt3a were measured by RT -PCR and Western Blot. Results At 28 days, there was no hydrocephalus in the normal control group and sham - IVH group. 80 %(12/15) of the rats in IVH group developed hydrocephalus. The rate was only 20 % ( 3/15 ) in deferoxamine - treated group, which was much lower than that in IVH group. The expression of Wnt1 mRNA was normal in normal control group and shame - IVH group, and upregulated in IVH group. The peak expression was detected at 28 days. The expression was significantly higher than that in shame - IVH group at 7 days and 28 days, after deferoxamine treatment, downregulation of Wnt1 mRNA were observed and were significantly lower than that in IVH group. The Wnt3a mRNA had similar trends, while the expressed level was normal in normal control group and shame - IVH group, and upregulated following IVH. The peak expression was detected at 28 days, and was significantly higher than that in shame - IVH group at 7 days and 28 days. After deferoxamine treatment, the downregulation of Wnt3a mRNA was observed and was significantly lower than that in IVH group. Accordingly, Wnt1 protein expression was normal in normal control group and shame - IVH group, and increased after IVH. At 7 days and 28 days, the Wnt1 protein expression of IVH group was markedly higher than shame - IVH group. After deferoxamine treatment, the expression of Wnt1 protein was reduced and significantly lower than that of IVH group. The expression of Wnt3a protein was also normal in normal control group and shame - IVH group. The up-regulation of the protein expression was observed following IVH at 28 days, while down - regulation was observed after deferoxamine treatment at 28 days. Conclusions Deferoxamine could reduce hydrocephalus after IVH, and Wnt pathway may play an important role in the development of IVH and therapeutic effect of deferoxamine.     

An experimental model to study isolated effects of thrombin in vivo

Background

In addition to a recognized role in the coagulation cascade and haemostasis, thrombin is known to have multiple functions. We hypothesized that protracted intravenous infusion of thrombin at steady state will allow to study isolated thrombin effects in vivo.

Methods

Thrombin (0.05-0.9 U/kg/min) was continuously infused in Sprague Dawley rats over five hours (n = 38). The study consisted of three parts: dose escalation (n = 21), dose verification (n = 5) and a parallel group study to investigate whether thrombin effects can be antagonised by concomitant infusion of lepirudin (n = 12).

Results

A thrombin dose of 0.9 U/kg/min decreased platelet counts by 70% compared to the control group (median 230 × 10^9/L vs. 752 × 10^9/L; p = 0.041). In accordance, infusion of 0.9 U/kg/min of thrombin decreased fibrinogen level by 75% compared to the control group (56 mg/dl vs. 220 mg/dl; p = 0.046). Cumulative thrombin doses of ≥ 0.1 U/kg/min caused bleedings but not thromboembolic events. Thrombin at doses ≥ 0.15 U/kg/min was lethal in four cases (30%). Platelet counts and fibrinogen levels after thrombin infusion correlated with bleeding events and mortality. Administration of thrombin at cumulative doses of 0.3-0.9 U/kg/min was associated with a 3 to 6.5 -fold increase in IL-6 levels (139-306 pg/ml vs. 47 pg/ml, p < 0.05). In contrast, thrombin infusion did not alter other markers of inflammation (IL-10, MCP-1 or TNF-alpha). In addition, lepirudin prevented thrombin- induced thrombocytopenia.

Conclusion

Protracted intravenous infusion of thrombin offers a new experimental model, where consumption of fibrinogen and platelets correlates with bleedings and mortality. Infusion of thrombin increased only IL-6 levels but not other cytokines.